Methods and processes for attaching compounds to matrices

ABSTRACT

The present invention describes extremely rapid and efficient methods for the attachment of chemical moieties to matrices by the use of microwave technology. The methods of the invention can be applied in a variety of ways for the preparation of different types of matrices for a variety of applications including but not limited to the functionalization of various solid supports, and matrices in the form of powder, beads, sheets, and other suitable surfaces for use in applications including but not limited to oligonucleotide synthesis, peptide synthesis, environmental clean up (removal of toxic materials), immunoassays, affinity chromatography, combinatorial chemistry, microarrays, proteomics and medical diagnostics.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/558,778, filed on Apr. 1, 2004, U.S. Provisional Application No.60/583,413, filed on Jun. 28, 2004 and U.S. Provisional Application No.60/616,388, filed on Oct. 6, 2004. The entire teachings of the aboveapplications are incorporated herein by reference.

GOVERNMENT SUPPORT

The invention was supported, in whole, or in part, by NIH grant number 5UO1 AI058270-02. The Government has certain rights in the invention.

FIELD OF THE INVENTION

This invention relates to novel methods of attaching chemical moietiesto inorganic and organic matrices. The matrices carrying such chemicalmoieties can be used in a number of applications such as in solid-phasesynthesis, diagnostic devices, biosensors, catalysts, scavengers, and indrug delivery systems.

BACKGROUND OF INVENTION

Since the first report of solid-phase synthesis of peptides byMerrifield in 1962, several applications of using solid matrices haveevolved over the past 50 years. For example, solid-phase synthesis isnow routinely employed for the synthesis and manufacture ofmacromolecules such as peptides, carbohydrates, and oligonucleotides.Also, several organic reactions are routinely performed in solutionphase employing reagents that are covalently bound to solid matrix.Following the reaction, the matrix containing the reagent is simplyfiltered off from the reaction medium enabling partial purification ofthe desired product from the solution. In another application, compoundsattached to solid matrices that carry acidic and basic moieties areemployed as “scavengers” of basic and acidic reagents respectively fromreaction media.

Several catalysts employed in organic synthesis are often employed assolid matrix. Compounds attached to solid matrices are also employed assensors in detection devices. In yet another application, drug moleculesattached to solid matrices are used as delivery systems for topical andsystemic administration of drugs.

Macromolecules such as oligonucleotides as antisense compounds and asagents of RNA interference are synthesized and manufactured routinelyusing solid-phase synthesis. In this case, the first nucleotidic oramino acid residue (also referred to as the leader building block) iscovalently attached to the solid matrix via a linker arm. The subsequentaddition of monomeric units to the leader block is carried out on thesolid matrix. Upon completion of the assembly of the macromolecule, thematrix is treated with a chemical reagent that cleaves the linker arm ofthe leader block thereby releasing the macromolecule into solution.

The attachment of chemical moiety to the matrix is a very important stepfor various applications of the matrices. Usually the chemical moiety isemployed as a solution, which is then contacted with the matrix with andwithout the aid of a catalyst or other reagent. Since this is a biphasicreaction involving solid and liquid matrix, reaction times can oftenvary from several hours even into days before complete reaction occurs.Consequently, the initial attachment of the chemical moiety to thematrix is the rate-limiting step. Furthermore, an important parameterfor the use of any matrix is the “loading of the support” (expressed asmicromol/g) with the chemical moiety. A low loading would necessitatethe use of larger amounts of the matrix to accomplish a desiredapplication objective. This results in substantial increase in cost. Theloading protocols employ hazardous solvents and reagents and takes longreaction times. The commonly employed loading processes are alsoinefficient since, often incomplete reaction results in “uncapped”functionalities on the solid matrix. Thus e.g., when a loaded matrix isemployed in solid-phase synthesis, uncapped functionalities on the solidmatrix would interfere with the subsequent synthetic steps therebydecreasing the overall yield of the desired product. Thus an additional“capping step” needs to be employed before the matrix can be used insynthesis.

Clearly, efficient processes for attaching chemical moieties todifferent types of matrices will be of great value in the use of thematrices for different applications.

SUMMARY OF INVENTION

The present invention describes extremely rapid and efficient methodsfor the attachment of chemical moieties to matrices by the use ofmicrowave technology. The methods of the invention can be applied in avariety of ways for the preparation of different types of matrices for avariety of applications including but not limited to thefunctionalization of various solid supports, and matrices in the form ofpowder, beads, sheets, and other suitable surfaces for use inapplications including but not limited to oligonucleotide synthesis,peptide synthesis, environmental clean up (removal of toxic materials),immunoassays, affinity chromatography, combinatorial chemistry,microarrays, proteomics and medical diagnostics.

DETAILED DESCRIPTION OF THE INVENTION

With the recent advances in genomics and proteomics, small molecules andmacromolecules have been discovered against disease targets and haveshown promise as potential drug candidates. However several analogs ofthese early stage compounds need to be synthesized and tested for leadoptimization. Most synthetic processes are slow and time consuming.Consequently, the advancement of early lead compounds by way ofsynthesis and testing is a slow process. The rapid synthesis and testingof many compounds will help accelerate lead optimization and drugdevelopment.

Most developments in chemistry have been concerned with highly reactivereagents in solution or solid phase. The energy for most chemicalreactions is provided by heat transfer equipment such as oil baths, sandbaths and heating jackets in contact with the reaction vessel. Thesemethods of heat transfer are inefficient resulting in slow andnon-uniform heating of reactants. In contrast, Microwave energy has beenfound to induce dramatic rate accelerations of chemical reactions.During the past 20 years, it has been found that a large number ofchemical reactions that took several hours and days to complete can bedone in less than five minutes through the intervention of microwaveenergy. This is because microwave radiation induces dielectric heatingand almost all the energy associated with it is used to directly heatthe reactants and solvents and not the reaction vessel. Microwaveradiation passes through the walls of the vessel and heats only thereactants and the solvent and not the reaction vessel itself. In aproperly designed apparatus, the temperature will be uniform throughoutthe reaction media and therefore there is less likelihood of byproductsand decomposition products.

If the reaction vessel is kept under pressure, rapid increases intemperature can result that are much higher than the boiling point ofthe solvent. The reason for this is two fold: microwave radiation hasboth an electric field and magnetic field component. The former isresponsible for heating. The heat is generated by rapid kinetic energy(rotational and vibrational energy) released by alignment of the dipolesof the dilelectic solvent with the applied electric field. Anotherfactor which results in heating is the conductance process due topresence of ions in the medium that increases the collision rateconverting the kinetic energy into heat. Consequently, to be effectivein the microwave process, the solvents that are employed should have ahigh dilectric constant. These solvents include, water, (dimethylsulfoxide (DMSO), DMF (Dimethyl formamide), dimethyl acetamide, N-methylpyrrolidone, acetonitrile (CH₃CN), Methanol, ethanol, acetone etc.However, the inherent hazard of violent explosions due to higherpressures and temperature developed in close vessel under microwaveradiation has led to modified protocols in some cases. Heavy-walledreaction chambers with pressure release systems and solvent-lessreactions have led to more and safer operations.

A number of reactions have been described in the literature that havesuccessfully utilized microwave processes. These include N-acylation,alkylation, nucleophilic substitutuion, aromatic substitution,cycloaddition, deprotection and protection of functional groups,esterification and transesterification, heterocycle synthesis,organometallic reactions, oxidations, rearrangements, and reductions.Several “name reactions” such as Diels-Alder, Knoevenagel condensations,Hech and Suzuki couplings etc. are included in the above list.(Lidstrom, P., Terry, J., Wathey, B., Westman, J. Tetrahedron, 2001, 57,9225).

It is pertinent to mention that microwave reactions have also beenemployed both in solution-phase and solid-phase reactions. For example,Larhed et al., (Larhed, M., Hallberg, A. J. Org. Chem. 1996, 61, 9582)used microwave to enable solid-phase Suzuki coupling reactions. Ugi“four-component” reactions have been done in solid-phase using microwave(Hoel, A. M. L., Nielsen, J. Tetrahedron Lett., 1999, 40, 3941-44).Heterocycles such as succinimide have been synthesized on anamine-terminated polystyrene resin upon reaction with substitutedsuccinic anhydrides in the presence of TaCl₃-SiO₂ catalysis(Chandrasekhar, S., Padmaja, M. B., Raza, A. Synlett. 1999, 10, 1597).Other examples include transesterifications of allyl alcohol teriminatedpolystyrene soluble polymer with acetoacetic ester (Vanden Eynde, J. J.,Rutot, D. Tetrahedron, 1999, 55, 2687), hydrothermal co-condensation onsolid support (Ganschow, M., Wark, M., Wohrle, D., Schulz-Ekloff, G.Angew Chem Intl. Ed. Engl. 2000, 1, 161. However, most reactions havebeen reported in solution-phase.

Although as stated above, microwave-assisted processes have beenemployed in solid-phase synthesis, there is no report ofmicrowave-assisted loading of building blocks on to solid supports.Several applications of the novel, microwave-assisted processes of theinvention are anticipated. For example, the functionalized solid-supportcan be readily loaded with nucleosides for use in oligonucleotidesynthesis in accordance with the invention. Starting from(un-derivatized), native controlled-pore-glass (CPG), completefunctionalization and loading of nucleosides can be done within 24 to 48hours ready for DNA synthesis using the methods of the invention. Theconventional procedures require between 7 to 10 days to obtain fullyloaded supports. Using the present invention, nucleoside-loaded supportscan be made cheaper, faster and more eco-friendly. Several 100 g batchesof the supports with consistent high loadings of 70 to 80 micromol/ghave been obtained as discussed in the Examples. Consequently, thisprocess will be very useful both for smaller scale, and larger-scaleoperations.

Another application of the invention relates to the rapid preparation offunctionalized matrices for applications in microarrays, proteomics,medical diagnostics, and the like. In yet another application of theinvention, rapid preparation of functionalized supports for loading withamino acids, nucleoside derivatives, sugars and other carbohydrates,small molecules, antibodies, proteins, amino acids, peptides, and otherligands and macromolecules, for a variety of applications includingpeptide and carbohydrate synthesis and environmental clean up (removalof toxic materials), radioimmunassays (RIAs), fluorescence immunoassays(FIAs), ELISA, and Affinity Chromatography is made possible.

In one embodiment, the invention provides a method for attaching achemical moiety to a matrix comprising the steps of: (a) contacting thematrix with a reagent capable of a nucleophilic group such as an amino,hydroxyl, or carboxylic group to the matrix; (b) exposing the reactionmixture of step (a) to microwave radiation thereby resulting in afunctionalized matrix; (c) contacting the functionalized matrix of step(b) with a reagent capable of forming an ester or amide bond with thematrix and further comprising free carboxyl termini on the matrix; (d)exposing the reaction mixture of step (c) to microwave radiation therebyforming a mono-ester or mono-amide linkage with the matrix comprisingfree carboxyl termini on the matrix; and (e) contacting the carboxylatedmatrix of step (d) with the chemical moiety via a functionalized regionof the chemical moiety capable of reacting with the carboxylated matrixthereby resulting in a matrix functionalized with the chemical moietysuch as amino, hydroxyl, or thiol groups.

Suitable matrices include but are not limited to, controlled pore glass(CPG), glass in the form of any glass surface natural or modifiedincluding beads or powders, silica gel, alumina, polystyrene, tentagel,polyethylene glycol, cellulose, Teflon, and their derivatives, ceramic,zeolite, clay, as well as, matrices such as, Ti, Carbon, Si, gold, orother metal surfaces.

In a preferred embodiment, steps (a) and steps (c) of the above methodare carried out in the presence of a solvent having a dielectricconstant. Suitable solvents having dielectric constants include but arenot limited to, dimethyl formamide, dimethyl acetamiide, N, N-dialkylformamides and acetamides, N-methyl pyrrolidone, DMSO, and alcoholsincluding polyols.

In a preferred embodiment, the exposing to microwave radiation steps of(b) and (d) comprises exposing the reaction mixtures to microwaveradiation for a total time of at least about 4 minutes. The exposingsteps may comprise pulsed radiation or constant microwave radiation.

For all embodiments described herein, suitable reagents capable ofadding a nucleophilic group include but are not limited to, substitutedand unsubstituted amines, alcohols and thiols.

For all embodiments described herein, suitable reagents capable offorming an ester or amide bond with the matrix and further comprisingfree carboxyl termini on the matrix such as substituted andunsubstituted amino acids, hydroxyl acids, thiol acids and halo acids.

For all relevant embodiments described herein, examples offunctionalized regions of the chemical moieties that are capable ofreacting with the carboxylated matrix include but are not limited toregions comprising,amino, hydroxyl, thiol, X—R reagents (where X ishalogen, R is alkyl, aryl, aralkyl, cycloaclkyl, heterocyclics.

For all relevant embodiments described herein, examples of chemicalmoieties suitable for attachment to the functionalized matrices preparedin accordance with the invention include but are not limited to,modified and unmodified nucleosides and nucleotides, DNA, RNA, modifiedand unmodified amino acids, peptides, proteins, synthetic blockpolymers, heterocycles, organometallic synthesis reagents, lipidssteroids and carbohydrates.

The term “nucleoside” as used herein refers to a heterocyclicnitrogenous base in N-glycosidic linkage with a sugar. Nucleosides arerecognized in the art to include natural bases (standard), and modifiedbases well known in the art. Such bases are generally located at the 1′position of a nucleoside sugar moiety. Nucleosides generally comprise abase and sugar group. The nucleosides can be unmodified or modified atthe sugar, and/or base moiety (also referred to interchangeably asnucleoside analogs, modified nucleosides, non-natural nucleosides,non-standard nucleosides and other; see for example, Usman andMcSwiggen, supra; Eckstein et al., International PCT Publication No. WO92/07065; Usman et al., International PCT Publication No. WO 93/15187;Uhlman & Peyman, supra, all are hereby incorporated by referenceherein). There are several examples of modified nucleic acid bases knownin the art as summarized by Limbach et al., 1994, Nucleic Acids Res. 22,2183. Some of the non-limiting examples of chemically modified and othernatural nucleic acid bases that can be introduced into nucleic acidsinclude, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl,pseudouracil, 2,4,6-trimethoxy benzene, 3-methyl uracil, dihydrouridine,naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5-methylcytidine),5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g.,5-bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.6-methyluridine), propyne, quesosine, 2-thiouridine, 4-thiouridine,wybutosine, wybutoxosine, 4-acetylcytidine,5-(carboxyhydroxymethyl)uridine,5′-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1-methyladenosine, 1 -methylinosine, 2,2-dimethylguanosine,3-methylcytidine, 2-methyladenosine, 2-methylguanosine,N6-methyladenosine, 7-methylguanosine,5-methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine,5-methylcarbonylmethyluridine, 5-methyloxyuridine,5-methyl-2-thiouridine, 2-methylthio-N-6-isopentenyladenosine,beta-D-mannosylqueosine, uridine-5-oxyacetic acid, 2-thiocytidine,threonine derivatives and others (Burgin et al., 1996, Biochemistry, 35,14090; Uhlman & Peyman, supra). By “modified bases” in this aspect ismeant nucleoside bases other than adenine, guanine, cytosine and uracilat 1′ position or their equivalents; such bases can be used at anyposition, for example, within the catalytic core of an enzymatic nucleicacid molecule and/or in the substrate-binding regions of the nucleicacid molecule.

The term nucleoside as used herein further includes “protectednucleosides”. A protected nucleoside has a protecting group, such as a5′dimethoxytrityl group. Preferably a 5′dimethoxytrityl protectednucleoside with a free (unprotected and uncapped) 3′hydroxyl group.Protecting groups are used to prevent undesirable side reactions withreactive groups present in the nucleoside thereby allowing selectivereaction at the desired location of the nucleoside or nucleotide ofinterest.

In one preferred embodiment, the invention provides a method for rapidlyattaching a chemical moiety to a matrix comprising the steps of: (a)contacting the matrix with a reagent capable of adding thiol ester, athioamide, a sulfonamide, a sulfonate ester a phosphoramide orphosphoric ester on the matrix; (b) exposing the reaction mixture ofstep (a) to microwave radiation thereby resulting in a functionalizedmatrix with free carboxy termini; (c) contacting the functionalizedmatrix of step (b) with a reagent capable of forming an ester or amidebond with the matrix and having a free carboxyl termini on the matrix;(d) exposing the reaction mixture of step (c) to microwave radiationthereby forming a mono-ester or mono-amide linkage with the matrixhaving free carboxyl termini; and (e) coupling the carboxylated matrixof step (d) with the chemical moiety via a functionalized region of thechemical moiety capable of reacting with the carboxylated matrix,thereby resulting in a matrix functionalized with the chemical moiety.

In yet another embodiment, the invention provides rapidfunctionalization of solid supports by microwave-assisted processes andtheir subsequent utilization in the loading of nucleosides. In thisembodiment (Scheme 1), controlled pore glass support (CPG) is contactedwith 3-aminopropyl-triethoxysilane, either neat or in presence ofdimethylformamide (DMF) as solvent under microwave conditions. Theresulting amino-functionalized CPG is then reacted with succinicanhydride in DMF under microwave conditions. The succinylated CPG isthen contacted with 5′-protected nucleoside derivative, preferably5′-dimethoxytrityl-protected nucleoside with a “free” 3′-hydroxyl group,either under microwave or without microwave conditions.

The abbreviations used in Scheme 1 are as follows: MORE,(microwave-induced organic reaction); DMAP, 4,4-dimethylaminopyridine;Py, Pyridine; DMF, Dimethylformamide; EDC, Ethyl3-(3-dimethylaminopropyl) carbodimide.

Thus, in a preferred embodiment, the invention provides a method forpreparing a functionalized matrix for oligonucleotide synthesiscomprising the steps of: (a) contacting the matrix with a reagentcapable of adding an amino functional group to the matrix; (b) exposingthe reaction mixture of step (a) to microwave radiation therebyresulting in an amino-functionalized matrix; (c) contacting theamino-functionalized matrix of step (b) with a succinylating reagentcapable of chemically succinylating the matrix; (d) exposing thereaction mixture of step (c) to microwave radiation thereby resulting ina succinylated matrix; (e) coupling the succinylated matrix with anucleoside derivative capable of reacting with the succinylated matrixthereby forming a functionalized matrix suitable for further use in thesynthesis of oligonucleotides and (f) optionally recovering excessnucleosides used step (e).

In one preferred embodiment, step (e) above, further comprises the useof a solvent such as DMF for coupling of nucleosides to the succinylatedmatrix.

In another preferred embodiment, step (f) is carried out by collectingthe filtrate from the reaction in step (e) and coupling excessnucleosides onto succinylated supports during aqueous work up of thefiltrate.

In all relevant embodiments described herein, examples of suitablesuccinylating reagents include but are not limited to succinicanhydride, substituted dicarboxylic acids such as substituted succinicacid, substituted glutaric acid and their corresponding anhydrides, or areagent capable of forming a mono-ester linkage with the matrix andhaving a free carboxyl termini group.

As used herein, a reagent capable of adding an amino functional group toa matrix is known as an “aminating reagent”. Suitable aminating reagentsuseful in all relevant embodiments of the invention include but are notlimited to, 3-amino-alkyl silane, 3 amino-propyl-dimethyl-ethoxy silane,or N-[3-triethoxysilyl)propyl]ethylene diamine.

In one embodiment, a matrix with a predetermined loading capacity (oftenlow-loaded supports) may be obtained by using a mixture of two reagents,one of which will serve as the aminating reagent and the other serves asthe filling agent (e.g. phenyltriethoxy silane). The reaction of thefiller agent (or capping reagent) with the nucleophilic groups on thesupport results in a modified support that lacks a reactive functionalterminus (such as an amino group). Consequently the filled or cappedsite on the matrix is not available for nucleoside derivatization. Atthe same time, the reaction of the aminating reagent with the supportresults in an amino-terminated support which can react with otherfunctional groups such as a carboxyl group.

The processes of the invention can be utilized without limitation forloading of nucleosides on a variety of commercially available matricessuch as controlled pore-glass (CPG), polystyrene, tentagel, polyethyleneglycol based (PEGA) supports and the like. Typically the reaction isperformed in industrial microwave equipment suitable for chemicalreactions or even a domestic microwave with the proper equipment. In oneembodiment of the invention, reactions are performed for 3 to 4 minutesin one stretch. In another embodiment of the invention, reactions areperformed in pulsed intervals of up to 30 seconds each followed by waitperiod of 5 minute. Preferably, the total microwave exposure time isabout 4 to 6 minutes.

Typical loadings of amino groups by microwave-assisted procedures inaccordance with the invention have ranged from 80 to 120 micromol/g.Nucleoside loadings of up to 90 micromole/g has been achieved inaccordance with the invention. These loadings are much higher thanloadings obtained by prior art protocols. Using prior art protocols, thenucleoside loadings only of 30 to 50 micromol/g are usually achieved.

In another embodiment of the invention, the loading capacity of aminogroups and nucleosides is enhanced by treating an amino-functionalizedCPG with a bifunctional reagent such as phenyldiisothiocyanate (Scheme2). This results in the formation of thiourea with a thioisocyanateterminus. Further reaction with polyamidoamine (PAMAM) yields CPG withmultiple amino sites on the surface. The CPG can then be succinylatedand loaded with nucleoside to get further ultrahigh loading.

In Scheme 2, R is a substituted or unsubstituted phenyl group or asubstituted or unsubstituted alkyl group.

Thus, in one embodiment, the invention provides a method of preparing afunctionalized matrix for oligonucleotide synthesis comprising the stepsof: (a) contacting the matrix with an aminoalkylsilane reagent in thepresence of a solvent having a dielectric constant and exposing thereaction mixture to microwave radiation thereby resulting in anamino-functionalized matrix; (b) reacting the amino-functionalizedmatrix of step (a) with a bifunctional reagent such asphenyldiisothiocyanate and exposing the reaction mixture to microwaveradiation in the presence of solvent thereby converting the amino groupson the matrix to thiourea groups whereby the matrix is furtherfunctionalized with a thioisocyanate terminus; (c) contacting the matrixof step (b) with polyamine such as polyamidoamine and exposing thereaction mixture to microwave radiation in the presence of a solventwith a dielectric constant thereby resulting in a matrix with multipleamino sites; (d) reacting the matrix of step (c) with succinic anhydridein the presence of dimethylformamide and exposing the reaction mixtureto microwave radiation thereby forming a succinylated matrix; and (e)coupling the succinylated matrix of step (d) with a 5′dimethoxytrityl-protected nucleoside derivative thereby forming afunctionalized matrix suitable for further use in oligonucleotidesynthesis.

In another embodiment of the invention (Scheme 3), native CPG is treatedwith bifunctional isocyanate to generate CPG with a carbamate orthiocarbamate linkage with a terminal isocyanate or thioisocyanatemoiety. Reaction with PAMAM generates CPG with multiple amino groups.

In Scheme 3, X is Oxygen or Sulfur and R is as previously defined inScheme 2.

Thus, the invention provides a method of preparing a functionalizedmatrix for oligonucleotide synthesis comprising the steps of: (a)contacting native CPG with bifunctional isocyanate or thioisocyanatereagent to generate CPG with a carbamate or thiocarbamate linkage with aterminal isocyanate or thioisocyanate moiety; (b) contacting the matrixfrom step (a) with polyamine such as polyamidoamine (PAMAM) to generatesCPG with multiple amino groups; (c) reacting the matrix of step (b) withsuccinic anhydride in the presence of dimethylformamide and exposing thereaction mixture to microwave radiation thereby forming a succinylatedmatrix; and(d) coupling the succinylated matrix of step (c) with a 5′dimethoxytrityl-protected nucleoside derivative thereby forming afunctionalized matrix suitable for further use in oligonucleotidesynthesis.

In another embodiment of the invention (Scheme 4), the native CPG istreated with activating groups such as p-nitrophenyl choloroformate, orcarbonyldimidazole, to form the activated groups amenable to furtherdisplacement by the PAMAM.

Thus, in another embodiment, the invention provides a method ofpreparing a functionalized matrix for oligonucleotide synthesiscomprising the steps of: (a) contacting native CPG with activatinggroups such as p-nitrophenyl choloroformate, carbonyldimidazole, etc toform the corresponding activated groups amenable to further displacementby amines and polyamines such as PAMAM; (b) reacting the matrix of step(a) with succinic anhydride in the presence of dimethylformamide andexposing the reaction mixture to microwave radiation thereby forming asuccinylated matrix; and (c) contacting the succinylated matrix of step(b) with a 5′ dimethoxytrityl-protected nucleoside derivative therebyforming a functionalized matrix suitable for further use inoligonucleotide synthesis.

In accordance with the invention, each reaction sequence is amenable tomicrowave conditions for rate enhancement and completion of eachindividual reaction sequences.

The methodologies described herein can be applied for functionalizationof other solid supports²³ and loading of supports with small molecules,nucleic acids, amino acids, peptides, proteins, antibodies,carbohydrates, sugars, and other macromolecules for uses in applicationsselected from: peptide and carbohydrate synthesis and environmentalclean up (removal of toxic materials), RIAs, FIAs, ELISA, and AffinityChromatography. A more detailed description of the many applications forfunctionalized supports may be found in U.S. Pat. No. 6,486,286,incorporated herein by reference in its entirety.

The approach described here can also be employed for rapidfunctionalization of other solid matrices in the form of beads, slides,or pins for application in microarrays (U.S. Pat. No. 6,486,286,incorporated herein by reference in its entirety), combinatorialchemistry including medical diagnostics, environmental clean up (removalof toxic materials), radio immunoassays, fluorescent immunoassays,ELISA, and affinity chromatography.

In yet another aspect, the invention provides a functionalized matrixfor oligonucleotide synthesis prepared by any one of the methodsdescribed herein. The invention further provides an oligonucleotideattached to a functionalized matrix prepared in accordance with any oneof the methods of the invention described herein.

The invention further provides methods for rapid deprotection andcleavage of oligonucleotide assembled on a solid support comprising thesteps of: a) taking up support-bound oligonucleotide in a heavy-walledcontainer with a stopper; (b) adding alkali such as NaOH of strength<0.2 N, but preferably 0.1 N NaOH; c) exposing the contents to microwaveradiation in 10 to 15 second cycles; d) maintaining outside temperatureof the container at 90 to 95° C. while heating and 75 to 80° C. whilecooling during each cycle; e) isolating the product by neutralizationand filtration; and (f) separating the support for recycling. In onepreferred embodiment, the separating step further comprises employingthe recycled support as matrix for rapid attachment of a chemical moietyselected from nucleic acids, proteins, antibodies, carbohydrates andother macromolecules for uses in applications selected from: peptide andcarbohydrate synthesis and environmental clean up (removal of toxicmaterials), RIAs, FIAs, ELISA, and Affinity Chromatography.

The invention is further illustrated by the following Examples.

EXAMPLES Example 1

Rapid and efficient functionalization of solid-support bymicrowave-assisted procedures has been achieved. The functionalizedsolid-support can be readily loaded with nucleosides for use inoligonucleotide synthesis. This method can also be extended to rapidfunctionalization of other solid matrices for application in microarraysand combinatorial chemistry.

Over the past two decades, microwave-assisted procedures have beensuccessfully employed in a number of synthetic transformations,resulting in rapid and efficient synthesis of different classes oforganic compounds.¹ Several advantages have been claimed in the use ofmicrowave-assisted organic synthesis²: (a) ultra fast reaction kinetics,(b) cleaner reactions with improved yields and reduced formation of sideproducts, (c) ability to effect, chemo-, regio-, and stereoselectivetransformations, (d) flexibility to perform reactions with or withoutsolvents, (e) economical and eco-friendly processes than thecorresponding conventional reactions, and (f) successful productformations in reactions that fail under conventional conditions.

Some microwave-assisted reactions pose significant risk of explosionespecially when conducted in closed vessels.² In such cases, reactionshave been conducted following deposition of reactants on solid matrixsuch as alumina, silica gel, and clay. Interestingly, the supports bythemselves were found to absorb very little microwave energy.³ Examplesof reactions carried out on matrix-bound reactants includetransesterfication,⁴ N-acylation,⁵ Ugi four-component condensation,⁶ andSuzuki coupling.⁷

However, there are only limited reports of application ofmicrowave-assisted procedures in the solid-phase synthesis of nucleicacids.¹ As is well-known, besides the actual assembly of apolynucleotide on solid support, there are two other critical steps innucleic acid synthesis⁸ (a) preparation of solid support (usuallycontrolled-pore-glass, [CPG]) loaded with the leader nucleoside, and (b)deprotection and cleavage of the assembled, support-boundoligonucleotide. Both steps involve prolonged, labor-intensiveoperations. Several improvements have been reported to facilitate rapiddeprotection and cleavage of oligonucleotides⁸ including a recent reportof microwave-assisted procedures.⁹ In contrast, there are only limitedreports of improved procedures for loading of nucleosides onfunctionalized supports and their utility has not been fully explored.

Reported methodologies¹⁰⁻¹² for functionalization and loading of CPG aretime-consuming, labor-intensive, and involve the use of toxic solvents.Scheme 5 shows the reaction sequences for functionalization and loadingof CPG¹³ and involve (Path A): (a) reaction of CPG with3-aminopropyltriethoxysilane to give the amino-functionalized CPG (b)reaction of aminated support 1 with succinic anhydride to givecarboxy-terminated CPG 2,¹⁴ and (c) reaction of 2 with nucleoside 3 togive the nucleoside-loaded CPG 4.

Alternatively, reaction sequence in Path B has also been employed, butis less convenient especially when modified nucleosides are used becauseof tedious purification to obtain hemi-succinylated intermediate 5.¹³Thus, starting from native CPG, complete set of sequence of reactionsrequire seven to 10 days to obtain nucleoside-loaded CPG 4.Consequently, availability of nucleoside-bound supports presents asignificant bottleneck in the synthesis and manufacture ofoligonucleotides.

We report here the use of microwave-assisted procedures for ultra fastfunctionalization of solid supports that enable rapid loading ofnucleosides on solid supports.

I. Microwave-Assisted Amination (MAA) of CPG

Using reported procedures,¹⁰⁻¹² we attempted the amination of CPG (500A, Prime Synthesis) by treatment with (3-aminopropyl)triethoxysilane(APTES) in toluene for 48 to 72 h, followed by capping of unreactedhydroxy groups with trimethylsilyl chloride. Referring to Scheme 5,although aminopropyl-CPG 1 could be isolated from the reaction, theheterogeneous amination reaction had to be performed in refluxingtoluene and was unmanageable. Also, on some occasions, after capping ofunreacted hydroxyl groups with trimethylsilyl chloride, the productisolated was found to be devoid of amino group. This prompted us toinvestigate MAA of CPG for the preparation of 1 using a domesticmicrowave oven (800 watts, High power setting).

For conducting MAA of CPG, (Scheme 5) a specially fabricatedheavy-walled glass chamber was employed. The chamber was fitted withTeflon screw cap with a chemically resistant o-ring. These microwavereactions should not be attempted in common laboratory glassware. Allmicrowave reactions should be carried out behind safety shields.Typically, the MAA of slurry of CPG (50 to 100 g) in APTES gave theaminated product 1 within a few minutes. Following filtration andwashing, aminated-CPG 1 with amino-loadings of 90 to 113 micromol/g wasobtained. Typically 3.5 mL APTES/g of CPG was employed. In all cases,the desired aminopropyl CPG was isolated after washings with toluene,methanol, dichloromethane and hexanes. Amino-loading was determined bystandard protocol¹³ by treating a known amount of support with DMTrCland Bu4N⁺ClO₄ and “trityl assay”¹³ was carried out on the resulting DMTrderivative. In order to ascertain if amino-loading of CPG could befurther increased, the initially formed 1 was subjected to MAA withAPTES. However, no further increase in loading resulted therebysuggesting that all available hydroxyl groups on the CPG had beenfunctionalized during the first reaction. Thus, amination of CPG wascompleted within a few minutes under microwave conditions.

We also evaluated the effect of different solvents on the MAA of CPGwith APTES, and the results are given in Table 1. TABLE 1 Effect ofsolvents on microwave-assisted amination of CPG Loading AminationReagent Solvent μmol/g APTES DMF 86 DMSO 116 Neat 113

Although MAA could be conducted in DMF and DMSO, we found that reactionusing slurry of CPG with APTES to be most convenient and alsoeco-friendly. Since APTES was used in excess in such a protocol, weattempted to use the recovered APTES in a subsequent MAA of CPG.However, MAA of CPG with recovered APTES was unsuccessful, probably dueto the contamination of APTES with the liberated ethanol.

In attempts to further increase the amino-loading of CPG, we carried outMAA reactions (all neat) in conjunction with other amination agents suchas (3-aminopropyl)trimethoxysilane (APTMS), andN-(3-trimethoxysilylpropyl)ethylenediamine (APTED) (Scheme 6).

Although the corresponding aminated CPG 1 and 7 were obtained, loadingwas not increased beyond that obtained with APTES. Without being held toany theory, it is believed that amination involves the condensation ofthree hydroxyl groups on the CPG matrix, with the three ethoxy groups ofAPTES to form 1. We rationalized that if each of the hydroxyl groups ofCPG could be engaged in reaction with a single molecule of amonoethoxysilane derivative, e.g. 3-aminopropyldimethylsilane (APDS),amino-CPG 6 with increased amino-loading could result. However, althoughMAA reaction of CPG with (3-aminopropyl)dimethylsilane (APDS) gave thecorresponding aminated product 6, the loading was not increased beyond96 micromol/g (Table 2). TABLE 2 Loading of CPG obtained usingmicrowave-assisted amination Amino- Reaction loading Amination reagentconditions micromol/g

Condition i, from FIG. 1 73

MAA, 5 min 98-113

MAA, 5 min 110 

MAA, 5 min 96

MAA, 5 min 79

In another experiment, following the first MAA with APDS, the resultingamino-CPG was further subjected to a second MAA using APTES, but therewas no further improvement in loading suggesting that all availablesites on the CPG had been functionalized at the first reaction itself asbefore.

We also evaluated the MAA of CPG with APTES using different additives(Table 3). TABLE 3 Effect of additives on microwave-assisted aminationof CPG Reagent Additive Loading (μmol/g) APTES BF₃.Et₂O 45 PTSA 128 TFA116 DMAP 110

Best results were obtained with p-toluene sulfonic acid and TFA giving 1with amino loadings of 128 and 116 micromol/g respectively. With borontrifluoride etherate as a catalyst, the product 1 had considerablyreduced loading (˜40 μmole/g).

During the MAA of CPG with APDS, in the presence of tin (IV) chloride asa catalyst, an explosive reaction resulted. It is interesting to notethat various metallic halides such as AlCl₃,¹⁵ CuBr,¹⁶ FeCl₃,¹⁷ BiCl₃,¹⁸ZnCl₂,^(19,20) InCl₃,²¹ and TaCl₃ ²² have been apparently safelyemployed in microwave-assisted synthetic reactions. Our results suggestthat additives and solvents influence the amino loading on CPG and hencethe final loading of nucleosides. Further investigation is in progressto prepare amino-CPG with a predetermined loading level.

II. Microwave-Assisted Succinylation (MAS) of Aminated CPG (1) withSuccinic Anhydride

Encouraged by the success in MAA of CPG, the MAS of the functionalizedsupport 1 (Scheme 5) were attempted under microwave conditions.Microwave-assisted reaction of 1 with succinic anhydride, without theaid of any solvent, resulted in a very dark yellow colored support,probably due to the formation of imide rather than the expected acid.Nevertheless, MAS of 1 (50 to 100 g scale) was achieved successfully togive 2, in the presence of catalytic amount of DMAP in DMF as solvent,in less than 5 min. Typically 0.4 g of succinic anhydride and 50 mg ofN,N-Dimethylaminopyridine each per one g of aminated CPG was employedfor MAS. Since this reaction was exothermic, each microwave exposure wascarried out in 30 sec cycles with intermittent cooling. After thereaction, the colored slurry was filtered, washed with dichloromethane,methanol, and hexanes, and dried. Similar results were obtained withTentagel and PEGA resins. Completion of reaction was ascertained bytesting for the absence of amino group on a sample of 1. The use of DMFin place of pyridine under microwave conditions makes MAS procedurehighly attractive for the preparation of succinylated CPG 2. In additionto DMF, the succinylation can also be carried out using DMSO,dimethylacetamide, or CH₃CN.

III. Coupling of Nucleoside to the Succinylated CPG (2):

With the succinylated support 2 in hand, we carried out the loading ofnucleosides using EDC, DMAP, and TEA (Scheme 5). When 2 was mixed with5′-O-DMT-deoxyadenosine (3, B=dA), DMAP, TEA, and EDC (in that order) inanhydrous pyridine and shaken in an orbital shaker, CPG 4 with highnucleoside loadings of 70 to 75 micromol/g was obtained. Similar resultswere obtained with 5′-O-DMT-T (3, B=T).

In separate experiments, we have found that anhydrous DMF could be usedinstead of pyridine for the loading of nucleosides on 2. The use of DMFprovides an eco-friendly alternative to pyridine. In addition, highloading of nucleoside (high-loaded CPG, 4) could be achieved whenloading was carried out in a specially fabricated reactor, mounted on arotary shaker, and with added provision for recycling of reactants (seecopending U.S. Ser. No. 60/574,465, 60/583,414, 60/626,597 and60/647,734) referred to herein as the “novel reactor.” Thus, using therecycling approach, significantly less molar excess of nucleoside couldbe used to achieve the same loading levels as compared to theconventional protocol. This could be due to efficient mixing of thesolid and liquid phases brought about by the rotary motion in thereactor in conjunction with the recycling process.

Microscopic examination revealed that microwave exposure did not affectthe porosity, particle size or other physical characteristics of theCPG. To confirm this, automated synthesis of polynucleotides (usingExpedite 8909 synthesizer) was carried out as below:

IV. Synthesis of Oligonucleotides:

The CPG-loaded nucleoside 4 prepared as above and the corresponding CPGprepared by conventional method¹⁰⁻¹³ was employed in the 10 micromolsynthesis of di- and 20-mer oligonucleotide (PO and PS) in an ExpediteSynthesizer using phosphoramidite chemistry.⁸ In both instances, thestepwise coupling yields was greater than 98% (as ascertained by tritylanalysis). Following the synthesis, each of the CPG was treated with 28%NH₄0H at 55 ° C. for 12 h to isolate the fully deprotected di-, andpolynucleotides. RP-HPLC analysis of crude mixture showed the profile ofcompounds prepared using both supports were similar.

In conclusion, a microwave assisted protocol for rapid and efficientfunctionalization of CPG has been achieved whereby CPG 2 carrying acarboxy-terminus could be obtained from native CPG within few hours, incontrast to the conventional procedures, which required several days.Efficient processes for loading of nucleosides on the resultingfunctionalized support 2 have also been achieved herein using anhydrousDMF as a solvent. Furthermore the use of a novel reactor in conjunctionwith recycling technology enabled efficient loading of the nucleoside onsupport.

REFERENCES

-   1. For a review see: Listrom, P.; Tierney, J.; Wathey, B.;    Westeman, J. Tetrahedron, 2001, 57, 9225-9283.-   2. For a review see: Bose, A. K.; Manhas, M. S.; Ganguly, S. N.;    Sharma, A. H.; Banik, B. K. Synthesis, 2002, No. 11, 1578-1591.-   3. (a) Loupy, A.; Petit, A.; Hamelin, J.; Texier-Boullet, F.;    Jacquault, P.; Mathe, D. Synthesis, 1998, No.9, 1213. (b) Chatti,    S.; Bortolussi, M.; Loupy, A. Tetrahedron, 2001, 57, 4365-70.-   4. Vanden Eynde, J. J.; Rutot, D. Tetrahedron, 1999, 55, 2687-2694.-   5. Yu, A. M.; Zhang, Z. P.; Yang, H. Z.; Zhang, C. X.; Liu, Z.    Synth. Commun. 1999, 29, 1595-1599.-   6. Hoel, A. M. L.; Nielsen, J. Tetrahedron Lett. 1999, 40,    3941-3944.-   7. Larhed, M.; Hallberg, A. J. Org. Chem. 1996, 61, 9582-9584.-   8. For a review see: Beaucage, S. L.; Iyer, R. P. Tetrahedron, 1992,    48, 2223-2311.-   9. Kumar, P.; Gupta, K. C. Nucl. Acids Res. 1997, 25, 5127-29.-   10. Majors, R.; Hopper, M. J. Chrom. Sci. 1974, 12, 767-778.-   11. Tundo, P.; Venturello, P. J. Am. Chem. Soc. 1979, 101, 660-6613.-   12. Matteucci, M. D. Caruthers, M. H. Tetrahedron Lett. 1980, 21,    719-722.-   13. Pon, R. T. In Attachments of Nucleosides to Solid-phase    Supports; Beaucage, S. L., Bergstrom, D. E., Glick, G. D.,    Jones, R. A. Current Protocols in Nucleic Acids Chemistry. John    Wiley; New York, 1999; pp 287-298.-   14. Damha, M. J.; Giannaris, P. A.; Zabarylo, S. V. Nucl. Acids Res.    1990, 18, 3813-21.-   15. Villemin, D.; Vlieghe, X. Sulfur Lett. 1998, 21, 199-203.-   16. Grigor'ev, A. D.; Dmitrieva, N. M.; El'tsov, A. V.; Ivanov, A.    S.; Panarina, A. E.; Sokolova, N. B. J. Gen. Chem. 1997, 67,    981-982.-   17. Vilemin, D.; Sauvaget, F. Synlett 1994, 435-436.-   18. Baruah, M.; Prajapati, D.; Sandhu, J. S. Synth. Commun. 1998,    28, 4157-4163.-   19. Garrigues, B.; Laurent, R.; Laporte, C.; Laporterie, A.;    Dubac, J. Liebigs Ann. 1996, 5, 743-744.-   20. Clarke, D. S.; Wood, R. Synth. Commun. 1996, 26, 1335-1340.-   21. Ranu, B. C.; Hajra, A.; Jana, U. Tetrahedron Lett. 2000, 41,    531-533.-   22. Chandrasekhar, S.; Padmaja, M. B.; Raza, A. Synlett 1999, 10,    1597-1599.-   23. Other solid supports such as Tentagel™ and    aminomethyl-polystyrene also could be functionalized using this    protocol. Unpublished results

Example 2

Amination of CPG:

In a pressure chamber equipped with a Teflon plug and a chemicallyresistant 0 ring (Chemraz), 3-aminopropyltriethoxysilane (APTES, 450 ml,‘3.5 ml/g) was added to Native CPG 500 CPG (1 35g) and this well mixedreaction mixture was heated in a house-hold microwave (800 watt) in 1min cycle for 8 mins. The contents of reaction mixture was mixed well byshaking between the heating cycles and allowed to cool intermittently.At the end of heating, the reaction mixture was allowed to cool to RT,filtered, washed with toluene (2×125 ml) followed by methanol (2×250ml), dichloromethane (2×250 ml) and finally with hexanes (2×250 ml).Washed sample was treated with drops of ninhydrin and heated and thepresence of amino group was indicated by strong purple color. Theaminated CPG was dried in a glass tray and a small sample was driedunder high vacuum overnight for amine loading by reported procedurethrough dimethoxytrityl content analysis as follows. Trityl analysis: To˜100 mg of dried amino CPG, added 1 ml each of DMTrCl (0.25M in DCM) andof Bu4N⁺ ClO₄ ⁻ (0.25 M). The mixture was mixed well in an orbitalshaker for 30-40 mins, filtered, washed with DCM (2×10 ml), MeOH (2×10ml) and again with DCM (2×10 ml). Finally this tritylated aminoCPGsample was dried under high vacuum for 3 hours at r.t. Amino loading wasdetermined following the reported protocols through trityl estimationand was found to be in the range between 100-115 micromol/g.

Succinylation of Aminopropyl CPG:

In a 500 ml pressure chamber with a Teflon screw cap stopper (with aChemraz 0 ring), aminopropylCPG (150 g) was taken followed by theaddition of a solution of succinic anhydride (SA, 60 g)4-Dimethylaminopyridine (DMAP, 6 g) in N,N-Dimethylformamide (DMF, 550ml). The reaction mixture was heated in the MW for 8-10 cycles of each30 sec duration. Dark colored reaction mixture was mixed well by shakingat the end of each cycle and allowed to cool between the heating cycles.At the end of heating cycles, the reaction mixture was allowed to coolto RT, a small sample was filtered, washed with methanol, DCM andhexanes (10 ml each). This sample supported was tested for thecompletion of succinylation by adding ninhydrin solution in ethanol andheating with heatgun. Absence purple color indicated the completion ofsuccinylation. If any trace purple color appeared, the heating cycle wascontinued for few more cycles and repeat the test by filtering a sample,washing and doing the ninhydrin test again.

After completion of reaction, the colored reaction mixture was filtered,washed with methanol (3×200 ml), DCM (3×200 ml), EtOAc (3×200 ml) andfinally with hexanes (3×200 ml.

Loading of Nucleoside on to Succinylated CPG:

Example: Coupling of 5′-DMT-N-bzdA to succinylated CPG

To succinylated CPG (65 g, 87 μmol/g amino loading) in a 1 lit singlenecked r.b. flask, was added freshly distilled anhydrous DMF (260 ml)(anhydrous DMF was prepared by distillation of dry DMF fromCaH2)followed by the addition of DMT-N-bzdA (18.7 g, 5 eq), DMAP (3.5g), TEA (4 ml) and finally EDC (5.47 g, 5 eq). The reaction flask wassealed with a rubber septa and mixed under orbital shaking (˜150 rpm)overnight. If the determined loading of a representative sample from thereaction was acceptable (>65 μmol/g), the reaction mixture was filtered,washed twice with methanol, DCM and hexanes (200 ml each) and dried inair overnight. The nucleoside loaded CPG was mixed under orbital shakingwith 325 ml each of CAP A and CAP B mixtures for 3 hours and the cappedsupport was filtered, washed twice with methanol, DCM and hexanes (300ml each). The loading of dried support was determined as 70-75 μmol/gand stored at 4° C.

Nucleoside 2: Coupling of 5′-DMT-dT to Succinylated CPG

To succinylated CPG (5g, 110 μmol/g amino loading) in a 100 ml singlenecked r.b. flask, added DMF (20 ml) followed by the addition of DMTdT(1.5 g, 5 eq), DMAP (0.35 g), TEA (0.5 ml) and finally EDC (0.52 g, 5eq). The contents of the flask were mixed under orbital shaking (˜150rpm) overnight. After the determination of initial loading (>65 μmol/g),the reaction mixture was filtered, washed twice with methanol, DCM andhexanes (20 ml each) and dried in air overnight. The nucleoside loadedCPG was mixed with 25 ml each of CAP A and CAP B mixtures for 3 hoursand the capped support was filtered, washed twice with methanol, DCM andhexanes (30 ml each). The loading of dried support was determined as 68μmol/g and stored at 4° C.

Recovery of Excess Unreacted Nucleoside: Recovery of DMT-NbzdA

The filtrate, after the isolation of nucleoside (DMT-NbzdA, example 1)loaded CPG, was slowly added to 10 volumes of ice-cold water containing2-5 g of sodium chloride. The solid separated was allowed to settle andfiltered. The solid was washed with water, extracted in chloroform andthe organic layer was washed repeatedly washed with 5% citric acid toremove any trapped DMAP, sodium carbonate solution (5%) and finallybrine. The chloroform layer was dried over sodium sulfate andconcentration gave the excess nucleoside in acceptable purity in %yield. The 1H-NMR of recovered DMT-N-bzdA was identical with thecommercial standard sample, obtained from Reliable Biopharmaceuticals(St. Louis, Mo.).

Recovery of CPG

The CPG obtained, after the isolation of SB9000, a dinucleotide analog,was heated with 0.1 N (10 ml/g) and heated at 48° C. overnight underorbital shaking. Cooled to RT, filtered, washed with water. To thisadded HCl (1N, 10 ml/g) and mixed at RT for 4 h in an orbital shaker.The solid support was filtered, washed twice with water, MeOH, ethylacetate and hexanes (each 5 ml/g). The obtained support was dried atroom temperature and the retention of integrity of the recycled supportwas established through the sequence of reactions amination,succinylation and nucleoside loading. The capped support, obtainedthrough recycling, has been effectively reused for repeated synthesis ofSB 9000.

Solid Support with Predetermined Nucleoside Loading Level

Example: Amination of CPG with a mixture of3-(aminopropyl)triethoxysiland (APTES) and Phenyltriethoxysilane (PTES)

1 g of CPG was mixed with APTES and PTES in different proportions andheated in a 5 or 10 ml pressure reactor for 6-7 min in 1 min cycles. Atthe end of heating the reaction mixture was filtered, washed thrice withmethanol, DCM and hexnes (10 ml each). The aminated products weresubjected to amino loading determinations. There is a strong indicationof a trend and the amino loading indeed affected by the ratio ofaminating reagent (APTES) and the filter (PTES). Details of differentexperiments are given below in Table 4. TABLE 4 Amino loading No. APTES(ml) PTES (ml) % APTES (by vol) (micromol/g) 1 1 1 50 98 2 1.5 1 60 92 32.0 1 66 90 4 2.5 1 71 98 5 3.0 1 75 97 6 1.0 2.0 33 59 7 0.5 2.0 20 60The aminated CPGs have been subjected succinylation reactions aspreviously described.

The patent and scientific literature referred to herein establishes theknowledge that is available to those with skill in the art. All UnitedStates patents and published or unpublished United States patentapplications cited herein are incorporated by reference. All publishedforeign patents and patent applications cited herein are herebyincorporated by reference. All other published references, documents,manuscripts and scientific literature cited herein are herebyincorporated by reference.

While this invention has been particularly shown and described withreferences to preferred embodiments thereof, it will be understood bythose skilled in the art that various changes in form and details may bemade therein without departing from the scope of the inventionencompassed by the appended claims.

1. A method for attaching a chemical moiety to a matrix comprising thesteps of: (a) contacting the matrix with a reagent capable of adding anucleophilic group; (b) exposing the reaction mixture of step (a) tomicrowave radiation thereby resulting in a functionalized matrix; (c)contacting the functionalized matrix of step (b) with a reagent capableof forming an ester or amide bond with the matrix and further comprisinga free carboxyl termini on the matrix; (d) exposing the reaction mixtureof step (c) to microwave radiation thereby forming a mono-ester ormono-amide linkage with the matrix comprising a free carboxyl termini onthe matrix; and (e) coupling the carboxylated matrix of step (d) withthe chemical moiety via a reative region of the chemical moiety capableof reacting with the carboxylated matrix thereby resulting in a matrixfunctionalized with the chemical moiety.
 2. The method of claim 1wherein the contacting of steps (a) and (c) are carried out in thepresence of a solvent having a dielectric constant.
 3. The method ofclaim 1 wherein the matrix is selected from the group consisting of:controlled pore glass; glass beads; glass powders; silica gels; alumina;substituted or unstubstituted polystyrene; polyethylene glycol;cellulose, ceramics, zeolite, clay, titanium (Ti), Carbon, silicon (Si),and gold.
 4. The method of claim 1 wherein the chemical moiety isselected from the group consisting of: modified and unmodifiednucleotides and nucleosides; DNA; RNA; amino acids; peptides; proteins;synthetic block polymers; small molecules; and organometallic synthesisreagents.
 5. A method for preparing a functionalized matrix foroligonucleotide synthesis comprising the steps of: (a) contacting thematrix with a reagent capable of adding an amino functional group to thematrix; (b) exposing the reaction mixture of step (a) to microwaveradiation thereby resulting in an amino-functionalized matrix; (c)contacting the amino-functionalized matrix of step (b) with asuccinylating reagent capable of chemically succinylating the matrix;(d) exposing the reaction mixture of step (c) to microwave radiationthereby resulting in a succinylated matrix; and (e) coupling thesuccinylated matrix with a nucleoside capable of reacting with thesuccinylated matrix thereby forming a functionalized matrix suitable forfurther use in the synthesis of oligonucleotides.
 6. The method of claim5 wherein the contacting of step (a) is carried out in the presence of asolvent having a dielectric constant.
 7. The method of claim 5 whereinthe reagent of step (a) capable of adding an amino functional group isan aminoalkylsilane.
 8. The method of claim 6 wherein the solvent isdimethylformamide, dimethyl acetamide, N,N-dialkyl formamides andacetamides, N-methyl pyrrolidone, and DMSO.
 9. The method of claim 5wherein the succinylating reagent is a substituted or unsubstituteddicarboxylic acid or their corresponding anhydrides, or any reagentcapable of forming a mono-ester linkage with the matrix and having afree carboxyl termini.
 10. The method of claim 9 wherein thesuccinylating reagent is succinic anhydride.
 11. The method of claim 5further comprising the step of recovering excess nucleoside generated inthe coupling step (e) by aqueous work up of the filtrate.
 12. The methodof claim 5 wherein the matrix is selected from the group consisting of:controlled pore glass; glass beads; glass powders; silica gels; alumina;substituted or unstubstituted polystyrene; polyethylene glycol;cellulose, ceramics, zeolite, clay, titanium (Ti), Carbon, silicon (Si),and gold.
 13. The method of claim of claim 5 wherein the matrix iscontrolled pore glass.
 14. The method of claim 5 wherein the nucleosidederivative is a 5′-protected nucleoside derivative.
 15. The method ofclaim 14 wherein the 5′ nucleoside derivative is5′-dimethoxytrityl-protected nucleoside with a free 3′ hydroxyl group.16. The method of claim 5 wherein the functionalized nucleoside matrixcomprises a loading of nucleoside derivative in the range of about60-100 micromoles of nucleoside derivative per gram of matrix.
 17. Themethod of claim 5 wherein the amino-functionalized matrix of step (b)comprises a loading of amino group in the range of about 60-120micromole of amino group per gram of matrix.
 18. The method of claim 5wherein the contacting of step (a) comprises contacting the matrix withat least two different reagents capable of adding amino functionalgroups to the matrix.
 19. A method of preparing a functionalized matrixfor oligonucleotide synthesis comprising the steps of: (a) contactingthe matrix with an aminoalkylsilane reagent in the presence of a solventhaving a dielectric constant and exposing the reaction mixture tomicrowave radiation thereby resulting in an amino-functionalized matrix;(b) reacting the matrix of step (a) with succinic anhydride in thepresence of dimethylformamide and exposing the reaction mixture tomicrowave radiation thereby forming a succinylated matrix; and (c)contacting the succinylated matrix of step (b) with a 5′dimethoxytrityl-protected nucleoside derivative thereby forming afunctionalized matrix suitable for further use in oligonucleotidesynthesis.
 20. A method of preparing a functionalized matrix foroligonucleotide synthesis comprising the steps of: (a) contacting thematrix with an aminoalkylsilane reagent in the presence of a solventhaving a dielectric constant and exposing the reaction mixture tomicrowave radiation thereby resulting in an amino-functionalized matrix;(b) reacting the amino-functionalized matrix of step (a) withphenyldiisothiocyanate and exposing the reaction mixture to microwaveradiation in the presence of solvent thereby converting the amino groupson the matrix to thiourea groups whereby the matrix is furtherfunctionalized with a thioisocyanate terminus; (c) contacting the matrixof step (b) with polyamidoamine and exposing the reaction mixture tomicrowave radiation in the presence of a solvent with a dielectricconstant thereby resulting in a matrix with multiple amino sites; (d)reacting the matrix of step (c) with succinic anhydride in the presenceof dimethylformamide and exposing the reaction mixture to microwaveradiation thereby forming a succinylated matrix; (e) contacting thesuccinylated matrix of step (d) with a 5′ dimethoxytrityl-protectednucleoside derivative thereby forming a functionalized matrix suitablefor further use in oligonucleotide synthesis.
 21. A method of preparinga functionalized matrix for oligonucleotide synthesis comprising thesteps of: (a) contacting native CPG with bifunctional isocyanate orthioisocyanate reagent to generate CPG with a carbamate or thiocarbamatelinkage with a terminal isocyanate or thioisocyanate moiety; (b)contacting the matrix from step (a) with polyamidoamine (PAMAM) togenerate CPG with multiple amino groups; (c) reacting the matrix of step(b) with succinic anhydride in the presence of dimethylformamide andexposing the reaction mixture to microwave radiation thereby forming asuccinylated matrix; and (d) contacting the succinylated matrix of step(c) with a 5′ dimethoxytrityl-protected nucleoside derivative therebyforming a functionalized matrix suitable for further use inoligonucleotide synthesis.
 22. A method of preparing a functionalizedmatrix for oligonucleotide synthesis comprising the steps of: (a)contacting native CPG with at least one activating group selected fromp-nitrophenyl, choloroformate, or carbonyldimidazole, to form thecorresponding activated groups amenable to further displacement byPAMAM; (b) reacting the matrix of step (a) with succinic anhydride inthe presence of dimethylformamide and exposing the reaction mixture tomicrowave radiation thereby forming a succinylated matrix; and (c)contacting the succinylated matrix of step (b) with a 5′dimethoxytrityl-protected nucleoside derivative thereby forming afunctionalized matrix suitable for further use in oligonucleotidesynthesis.
 23. A functionalized matrix for oligonucleotide synthesisprepared by a process comprising the steps of: (a) contacting the matrixwith a reagent comprising an amino group capable of functionalizing thematrix; (b) exposing the matrix and the reagent comprising an aminogroup to microwave radiation thereby resulting in anamino-functionalized matrix; (c) contacting the amino-functionalizedmatrix with a succinylating reagent capable of chemically succinylatingthe matrix; (d) exposing the amino-functionalized matrix and thesuccinylating reagent to microwave radiation thereby resulting in asuccinylated matrix; and (e) contacting the succinylated matrix with anucleoside capable of reacting with the succinylated matrix therebyforming a functionalized matrix suitable for further use in thesynthesis of oligonucleotides.
 24. An oligonucleotide attached to thefunctionalized matrix of claim
 23. 25. The method of claim 1 wherein thereaction mixtures of steps (b) and (d) are exposed to microwaveradiation for a total time of at least about 4 minutes
 26. The method ofclaim 5 wherein the reaction mixtures of steps (b) and (d) are exposedto microwave radiation for a total time of at least about 4 minutes. 27.The method of claim 21 wherein the reaction mixtures of step (a), (b),(c) and (d) are exposed to microwave radiation for a total time of atleast about 4 minutes.
 28. The functionalized matrix of claim 23 whereinthe reaction mixtures of steps (b) and (d) are exposed to microwaveradiation for a total time of at least about 4 minutes.
 29. A method forattaching a chemical moiety to a matrix comprising the steps of: (a)contacting the matrix with a reagent capable of adding thiol ester, athioamide a sulfonamide a sulfonate ester a phosphoramide or phosphoricester on the matrix; (b) exposing the reaction mixture of step (a) tomicrowave radiation thereby resulting in a functionalized matrix with atleast one free carboxyl termini; (c) contacting the functionalizedmatrix of step (b) with a reagent capable of forming an ester or amidebond with the matrix and having a free carboxyl termini on the matrix;(d) exposing the reaction mixture of step (c) to microwave radiationthereby forming a mono-ester or mono-amide linkage with the matrixhaving free carboxyl termini; and (e) contacting the carboxylated matrixof step (d) with the chemical moiety via a functionalized region of thechemical moiety capable of reacting with the carboxylated matrix,thereby resulting in a matrix functionalized with the chemical moiety.30. A method for rapid deprotection and cleavage of oligonucleotideassembled on a solid support comprising the steps of: (a) taking upsupport-bound oligonucleotide in a heavy-walled container with astopper; (b) adding alkali such as NaOH of strength <0.2 N, butpreferably 0.1 N NaOH in; (c) exposing the contents to microwaveradiation in 10 to 15 second cycles; (d) maintaining outside temperatureof the container at 90 to 95° C. while heating and 75 to 80° C. whilecooling during each cycle; (e) isolating the product by neutralizationand filtration; and (f) separating the support for recycling;
 31. Themethod of claim 30 wherein the separating step (f) further comprisesemploying the recycled support as matrix for rapid attachment of achemical moiety selected from nucleic acids, proteins, antibodies,carbohydrates and other macromolecules for uses in applications selectedfrom: peptide and carbohydrate synthesis and environmental clean up(removal of toxic materials), RIAs, FIAs, ELISA, and AffinityChromatography.